Longistatin, a novel plasminogen activator from vector ticks, is resistant to plasminogen activator inhibitor-1.

Thrombo-occlusive diseases are major causes of morbidity and mortality, and tissue-type plasminogen activator (t-PA) is recommended for the treatment of the maladies. However, both t-PA and u-PA are rapidly inactivated by plasminogen activator inhibitor-1 (PAI-1). Here, we show that longistatin, a novel plasminogen activator isolated from the ixodid tick, Haemaphysalis longicornis is resistant to PAI-1. Longistatin was relatively less susceptible to the inhibitory effect of SDS-treated platelet lysate than physiologic PAs. Platelet lysate inhibited t-PA and tcu-PA with the IC(50) of 7.7 and 9.1 µg/ml, respectively, whereas for longistatin inhibition IC(50) was 20.1 µg/ml (p<0.01). Similarly, activated PAI-1 (20 nM) inhibited only 21.47% activity of longistatin but almost completely inhibited t-PA (99.17%) and tcu-PA (96.84%). Interestingly, longistatin retained 76.73% initial activity even after 3h of incubation with 20 nM of PAI-1. IC(50) of PAI-1 during longistatin inhibition was 88.3 nM while it was 3.9 and 3.2 nM in t-PA and tcu-PA inhibition, respectively. Longistatin completely hydrolyzed fibrin clot by activating plasminogen efficiently in the presence of 20 nM of PAI-1. Importantly, unlike t-PA, longistatin did not form complex with PAI-1. Collectively, our results suggest that longistatin is resistant to PAI-1 and maybe an interesting tool for the development of a PAI-1 resistant effective thrombolytic agent.

Characterization of asparaginyl endopeptidase, legumain induced by blood feeding in the ixodid tick Haemaphysalis longicornis.

We characterize here a cDNA from the ixodid tick Haemaphysalis longicornis, which encodes an asparaginyl endopeptidase, legumain (HlLgm), that was present as a functional molecule in the midgut of this tick. Endogenous HlLgm was detected as a 38-kDa antigen in H. longicornis extracts and was seen throughout all developmental stages. Endogenous HlLgm was mainly localized in the midgut epithelium by immunohistochemistry, and was shown to be up-regulated by the host blood-feeding process. Recombinant HlLgm (rHlLgm) produced in Escherichia coli was shown to hydrolyze the synthetic substrate Z-Ala-Ala-Asn-MCA at the rate of 6.42x10(-4)mumol/min/mg protein. Its activity was inhibited by the thiol blocking reagents iodoacetamide and N-ethylmaleimide. The enzyme was shown to possess a unique feature of having an autocatalyzed cleavage at asparagines(364-365) at the C-terminus of both endogenous HlLgm and rHlLgm. rHlLgm degraded bovine hemoglobin and bovine serum albumin (BSA) showing its strict specificity for hydrolysis of the peptide on the carboxyl side of the asparagines, as demonstrated by internal amino acid sequence analysis of proteolytic product of BSA cleavage. These results suggest that HlLgm plays an important role in host blood-meal digestion and may be critical for the final process of digestion of blood components.